JP2003523320A - Methods for rapid PEG modification of viral vectors, compositions for enhanced gene transduction, compositions with enhanced physical stability, and uses therefor - Google Patents

Abstract

  (57) [Summary] Describes a method for rapid modification of viral capsid or envelope proteins using polyethylene glycol (PEG). Also provided are methods of delivering molecules using the PEG-modified viruses of the present invention. Compositions containing the PEG-modified virus of the invention are characterized by improved gene expression, reduced neutralizing antibodies and CTL production. Also provided is a virus composition having enhanced physical stability, wherein the virus is lyophilized in a formulation having a 1: 1 ratio of sucrose and mannitol.

Description

Detailed Description of the Invention

This study was conducted on NIH [P30 DK47757-05, -07 and PO1H.
L59407-02] and NIH / NIAMS [P01 AR / NS436.
48-04], partially funded. The US Government may have certain rights in this invention.

FIELD OF THE INVENTION The present invention relates generally to the field of gene delivery to host cells, and more specifically to viral vector-mediated gene delivery.

BACKGROUND OF THE INVENTION Viral-mediated gene delivery has been described for the delivery of therapeutic genes to patients. One limitation of currently known methods is the production of neutralizing antibodies (NABs) by the patient's immune response to the viral capsid, which inhibits significant levels of gene expression upon readministration. Therefore, there is a need for gene delivery methods that avoid these immune responses.

Covalent modification of proteins and enzymes with functionalized poly (ethylene) glycol (PEG) has been investigated. PEG is an uncharged hydrophilic linear polymer that is non-immunogenic and has very low order toxicity. Recently O'R
iordan et al. developed a method for covalently linking various polyethylene glycols to adenovirus capsid proteins [O'Riordan et al., Hu Gene Therapy , 10 : 1349-1358 (1999)]. However, this method requires incubation for a period of 20 to 24 hours.

What is needed is a viral vector that circumvents the limitations of current constructs for readministration, as well as a method for rapid production of high levels of such constructs.

SUMMARY OF THE INVENTION In one aspect, the invention provides a method of conjugating a recombinant virus to polyethylene glycol to enhance its transduction efficiency. The method involves activation of activated P
Reacting EG and recombinant virus for about 15 minutes to about 2 hours at room temperature; and stopping the reaction, thereby obtaining the PEG-conjugated virus. Most suitably, activated PEG and recombinant virus are reacted at a polyethylene glycol to virus ratio of about 10: 1. Desirably,
Recombinant virus is present at a concentration of about 1 × 10 ^{10 to} about 1 × 10 ¹⁵ particles per ml of reaction solution.

In another aspect, the invention provides a PEG-conjugated virus produced by the method of the invention.

In yet another aspect, the present invention provides a method for increasing transduction efficiency of a recombinant virus, which requires delivery of the modified recombinant virus of the present invention to a host cell.

[0009] In yet another aspect, the invention provides a method of re-administering a molecule to a selected host cell via a viral vector. The method comprises contacting a host cell with a PEG-modified virus of the invention (the virus comprising a molecule for delivery to the host cell); and a recombinant virus comprising the molecule. A step of contacting the host cell is required.

In yet a further aspect, the present invention provides compositions useful for delivering selected molecules to host cells. The composition comprises a PEG-conjugated virus produced by the method of the invention and a physiologically acceptable carrier.

In yet a further aspect, the invention provides a composition that enhances the physical stability of a viral vector. The composition comprises a recombinant viral vector comprising a molecule for delivery to a host cell, sucrose and mannitol, wherein the sucrose to mannitol ratio is about 1 to about 1. Desirably, the composition is about 1.2.
Lyophilized to a final moisture content of from about 1.7% to about 1.7%.

Other aspects and advantages of the invention will be apparent from the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel and rapid method for modifying proteins or enzymes with activated polyethylene glycol (PEG) polymers. Advantageously, the modified methods described herein are completed within a short period of time (eg, about 1-2 hours), occur under mild conditions (eg, room temperature, physiological pH), and in extreme conditions. Improves physical stability under storage conditions.

The method has been found to be particularly well suited for modifying the capsid protein of viral vectors. Advantageously, substantial gene expression is achieved using the PEG-modified viral vectors of the present invention upon intravenous and intramuscular readministration without compromising the recipient's immune system against other pathogens. Has been obtained.
The vectors of the present invention are also useful for delivery by other routes. The modified vectors of the present invention are particularly useful in therapeutics that require chronic treatment. further,
These modified vectors, with high titers of neutralizing antibodies, allow for significant levels of gene expression in patients. In addition, readministration can occur with the native virus after pre-exposure to the modified virus.

Therefore, the PEG modification method of the present invention is particularly well suited to the modification of recombinant viruses that contain the desired molecule for delivery to a host cell within a capsid or envelope protein. In a particularly desirable embodiment, the molecule for delivery is
A transgene (tra) under the control of regulatory sequences that direct its expression in a host cell.
nucleic acid sequence encoding nsgene). In another desirable embodiment, the molecule for delivery is a protein, chemical, enzyme or other moiety. The choice of molecule for delivery is not a limitation of the present invention.

In a particularly desirable embodiment, the method of the invention is used with an adeno-associated virus. However, these methods can be readily applied to other viruses that contain their genetic material within the capsid or envelope proteins. Examples of other suitable viruses with capsids or envelopes suitable for PEG modification include, inter alia, adenovirus, retroviruses, lentiviruses. For convenience throughout the specification, reference may be made to capsid proteins. However, it will be appreciated that similar methods may be applied to modify the viral envelope proteins. Similarly, the methods of the invention can be readily used to modify other proteins and enzymes, whether viral or non-viral. Polyethylene glycol, as defined herein (PEG) has the _{formula: H- (OCH 2 CH 2)} n
It is a polymer composed of repeating units of —OH, where n is from 2 to about 1000. Most suitably, the present invention is about 500 to about 500,000, about 1
PEG compounds having a molecular weight in the range of 000 to about 200,000 and about 5,000 to 100,000 are used. Substituted PEG compounds are included within this definition. In a presently preferred embodiment, substituted PEG
Is a monomethyoxy polyethylene glycol having a molecular weight of 5000 (MPEG
). MPEG formula CH ₃ - has a _{(OCH 2 CH 2) n -OH} , and whether or allowed produced by conventional synthesis techniques, or commercially may be purchased. Other suitable substituted PEG compounds can be readily determined or designed, inter alia, to present suitable chemical groups for activation and / or as regards their attachment to the viral protein capsid.

MPEG (or another selected PEG) is chemically activated prior to binding to a selected protein (eg viral capsid) or enzyme. Chemical activation may be performed using conventional techniques. Activated MPEG may be purchased from a variety of commercial sources. For example, Tresyl-MPEG (TMPEG)
), Succinimidyl succinate-MPEG (SSMPEG) and cyanuric chloride-MPEG (CCMPEG), Sigma Chemicals (Sigma Chemi).
cals) (St. Louis, Missouri). Alternatively, MPEG
The following scheme:

[0018]

[Chemical 1]

[0019] May be activated with a succinimidyl activated ester according to.

[0020]   MPEG has the following scheme:

[0021]

[Chemical 2]

[0022] May be activated with tresyl chloride according to.

MPEG may be activated with cyanuric chloride according to one of two schemes:

[0024]

[Chemical 3]

Another suitable scheme for activation of MPEG with cyanuric chloride is as follows:

[0026]

[Chemical 4]

Regardless of the activation compound or method utilized, the activated MPEG is then processed by prior art methods [eg Delgaldo, Biotechnol. Appl. Biochem. , 12 : 119-128 (1999)], using a method of the invention carried out under conditions that are more rapid and less severe than selected proteins (eg viral capsids). Combine with. More specifically, methods in the art require incubation for a period of 20 to 24 hours, and in some cases, varying concentrations of activity at regular intervals during the incubation period. Stepwise addition of PEGylated was required. The resulting product is "super-pegylated.
ted) virion ". Advantageously, the method of the present invention uses activated PEG
Eliminates this requirement for addition at regular intervals of and provides a PEG-modified product in about 1-2 hours.

[0028]   Proteins selected for modification by the method of the invention (eg viral caps
Is preferably purified from any contaminants. For example, a virus vector
Is a method known to those skilled in the art [eg K. J. Fisher et al.J. Virol, 70 : 520-532 (1996)].
. The viral vector is then purified, preferably by conventional methods. For example
, Cesium chloride (CsCl) gradient [K. J. Fisher et al.Nat. Med. ,Three: 306-312 (1997)] and the method of column chromatography.
Especially desirable. Various columns useful for purification are commercially available (eg,
For example, Bio-Rad, Perceptive Biosystems (Pe)
rseptive Biosystems)) and other methods are described in the literature.
Stated. The choice of purification method is not a limitation of the present invention.

Suitably, the construct selected for PEG modification (eg purified viral vector) is desalted to remove any residual salts from the purification process (eg CsCl) or storage medium. In a desirable embodiment, this desalting is performed on a commercially available chromatography column. After desalting, the viral vector is equilibrated with a buffer compatible with the PEG conjugation reaction described below, or a formulation that enhances viral stability during long term storage. Suitably, the vector may be equilibrated with the buffer chosen for use in the binding reaction. The buffer may be chosen taking into account convenience and factors such as the type of activated PEG used in the reaction.

For example, where the construct is to bind to TMPEG according to the present invention, a particularly desirable buffer is potassium phosphate buffer (KPBS) at a pH of about 7 to about 8, and most preferably 7.4. is there. For binding to SS MPEG and CC MPEG, purified protein (eg, band from CsCl gradient) is treated with sodium phosphate (pH about 6.5 to about 7.5, and most preferably 7.
2) and in sodium tetraborate (pH about 8.5 to about 9.9, and most preferably 9.2) buffer. However, given these parameters, one of ordinary skill in the art may adjust these buffers as needed.

Once the protein has been desalted (if necessary) and equilibrated with the selected buffer, the selected activated PEG is combined with the virus to form a reaction mixture of PEG and protein, where Activated PEG is 1: 1 to 20: 1
, And PEG to virus ratios ranging from about 5: 1 to 15: 1. A ratio of 10: 1 is preferred when the virus is rAAV. However, one of ordinary skill in the art can easily adjust these ratios as desired. Suitably, the reaction mixture is carried out for about 15 minutes to about 120 minutes with gentle stirring at room temperature (eg about 22 ° C to 24 ° C) and the reaction is stopped. One desired method of stopping the reaction is P
By adding excess L-lysine with respect to the amount of EG. An about 10-fold excess has been found to be desirable; however, one of ordinary skill in the art can readily select another suitable amount. Alternatively, the reaction may be stopped by lowering the temperature of the reaction to about 4 ° C. Desirably, the reaction is allowed to proceed until complete (ie, about 100%) modification of the target protein (eg, viral capsid) is achieved. However, in some circumstances, the reaction may stop when the modification is less than complete. For example, when utilizing certain activated CCPEGs, the modification is approximately 70% complete to avoid the aggregation observed with certain types of activated CCPEGs in certain proteins. In some cases it may be desirable to stop the reaction. However, there are ten advantages of the present invention, including increased transduction efficiency.
Reactions were 70% to 98%, 75% to 95%, 80% to 90% for a variety of reasons that would be readily apparent to one of skill in the art, since less than 0% modification has been observed.
Alternatively, it may be desirable to stop at 85% completion. The extent of modification can be determined using conventional methods. See, for example, the fluorescamine assay described in Example 3 herein.

The resulting PEG-modified protein is separated from unreacted PEG, excess lysine and reaction by-products. An example of a suitable separation method is CsCl centrifugation,
Alternatively, passing the reaction mixture through a column equilibrated with a suitable buffer (eg, 10 mM potassium buffered saline (KPBS) at pH 7.4). However, other separation methods can be easily selected. The fractions containing the PEG modified construct can then be confirmed by UV spectrophotometric analysis at 260 nm or other suitable method. After separation (purification), the PEG-modified construct (eg, virus) may be suspended in a solution suitable for storage and / or delivery to host cells.

Advantageously, the inventors have found that viruses modified by this method retain infectivity. Most suitably, the PEG-modified virus of the invention avoids the incorporation of activating groups into the modified virus, thus avoiding any problems associated with the potential immunogenicity of the binding moiety. To do. Accordingly, the invention further provides compositions comprising a PEG-modified virus of the invention and a carrier suitable for delivery of the composition to host cells for a variety of therapeutic and other purposes. Compositions of the invention A. PEG-Modified Constructs In one aspect, the composition of the invention comprises a PEG-modified protein or enzyme of the invention and a physiologically compatible carrier. In one particularly desirable aspect, the invention provides a composition containing a PEG-modified virus. Suitably, such a virus encodes a transgene under the control of regulatory sequences which direct its expression in host cells. Alternatively, the virus may carry another molecule that it is desired to deliver to the host cell.

A suitable dose of a PEG-modified virus is readily determined by one of ordinary skill in the art depending on the condition being treated, the health of the domestic or human patient, the age and weight, and other relevant factors. be able to. However, in general, a suitable dose may be in the range of 10 ¹⁰ to 10 ¹⁸ , and preferably about 10 ¹⁴ to 10 ¹⁶ viral particles per dose for an adult human having a weight of about 80 kg. This dose may be suspended in about 0.01 mL to about 1 mL of a physiologically compatible carrier and delivered by any suitable means. The dose may be repeated as needed or desired using any suitable delivery means, daily, weekly, monthly or at other selected intervals.

Suitably, such carriers are physiologically compatible, eg physiological saline, distilled water, phosphate buffered saline (PBS), suitable for administration to human or non-human mammalian patients. , Potassium (K) PBS, sodium PBS and the like. Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. The choice of carrier is not a limitation of the present invention.

In some cases, the composition of the invention comprises, in addition to the PEG-modified virus and carrier (s), other conventional pharmaceutical ingredients such as preservatives, chemical stabilizers. Or a surfactant may be included in the formulation. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol and parachlorophenol. For example, suitable chemical stabilizers may include gelatin and albumin. Optionally, the compositions of the invention may contain other components such as free amino acids, eg adenosine, carbohydrates, surfactants and other stabilizers. B. Formulations that Enhance Physical Stability In another aspect, the invention provides novel formulations that enhance the physical stability of viral vectors even under harsh storage conditions. In one aspect, these viral vectors are modified by the methods of the invention. However, the formulations described herein are useful for a variety of other viral vectors, which can be readily selected by one of ordinary skill in the art. In addition, the formulations described herein are desirable for a variety of non-viral vectors, such as plasmids, or other DNA or proteins that will be subject to lyophilization, long-term storage under freezer or refrigerated conditions, and shipping. be able to.

Suitably, for long-term storage, the viral vector is present in the formulation in a range of about 1.2% to 1.7%, and more preferably about 1.3% to 1.4% final. Lyophilized to contain the water content. Desirably, the formulations of the present invention contain a minimum of about 5 × 10 ^{10 to} about 1 × 10 ¹⁶ or about 5 × 10 ¹¹ viral particles per ml. When utilizing non-viral constructs, one of ordinary skill in the art can readily determine the appropriate concentration of the selected construct. For example, the formulations of the present invention have about 0
． 1 μg to about 10 mg of DNA, more preferably about 10 μg to about 1 mg
Or about 0.1 μg to about 10 mg of protein, more preferably about 10 μg to about 1 mg of protein. However, other suitable concentrations can be readily selected by one of ordinary skill in the art. Using these parameters, lyophilization may be performed using conventional techniques. Generally, Samb
look et al., Molecular Cloning: A Laboratory.
Manual, Cold Spring Harbor Laboratories (Cold
See Spring Harbor Laboratories, Cold Spring Harbor, NY.

In one particularly desirable embodiment, the construct (to be formulated into the composition of the invention (
(Eg viral vectors) are combined in solution with sucrose and mannitol. For viral vectors, a sucrose to mannitol ratio of about 1: 1 is particularly desirable. However, the sucrose to mannitol ratio may be adjusted to take into account the stability of the selected construct upon storage, and more desirably about 3: 4 sucrose to mannitol. For example, adeno-associated viral vectors generally tend to exhibit better stability than adenoviral vectors under long term storage conditions. For example, the ratio ranges from about 4: 1 sucrose to mannitol to about 1: 4 sucrose to mannitol, and more preferably about 3: 4 sucrose to mannitol, and most preferably about 1: 1. Good over.

Most preferably, the composition of the present invention having enhanced storage stability is about 0.5.
% To 5% β-cyclodextrin (BCD), and most preferably also the tertiary amine BCD. In one particularly desirable embodiment, the composition further contains about 0.5 mg / ml to about 1 mg / ml pluronic protamine.

Optionally, the composition of the invention may contain conventional pharmaceutical ingredients such as preservatives, carbohydrates, stabilizers or surfactants, such as those mentioned above.

The PEG-modified constructs of the present invention (whether formulated as described above or not) and / or formulated for long-term storage (using PEG-modified constructs). The compositions of the present invention, including those produced by or other constructs) may be delivered using methods known to those of skill in the art. If the PEG-modified vector (or other construct) is lyophilized, the vector can be easily reconstituted using methods known to those of skill in the art. For example, L. Rey and J. C. May, "Freeze-dry"
ing / lyophilization of pharmaceutical
and biological products ", Drugs and the Pharmaceutical Sciences , Vol. 96, 1.
999, Marcel Dekker: New York, NY; J. Pickal, "Freeze-drying of P"
Rotins ", Part 1: Process Design., Biop harm , September 1990: p . 18-27; MJ Pikal, Bioph arm. , 3 (8): 28-31 (1990). Methods of the Invention The present invention provides a method of delivering a transgene or other molecule to a human or domestic patient by delivering the PEG-modified construct to the patient. May be transduced in vivo or ex vivo, taking into account factors such as the selection of the pathogen and the condition for which the patient is being treated. ) Delivery. However, these methods May also be used to deliver other PEG-modified constructs, and other compositions of the invention A. In Vivo For in vivo delivery of the transgene, the target organ, Any suitable route of administration may be used, including direct delivery to a tissue or site, intranasal, intravenous, intramuscular, subcutaneous, intradermal, vaginal, rectal and oral administration. They may be combined within the course of treatment or immunization.

Suitable doses of PEG-modified virus are readily determined by one of ordinary skill in the art depending on the condition being treated, the health of the domestic or human patient, age and weight, and other relevant factors. be able to. However, in general, a suitable dose may be in the range of 10 ³ to 10 ¹⁸ , preferably about 10 ⁵ to 10 ¹⁶ particles per dose for an adult having a weight of about 80 kg. This dose is formulated into a pharmaceutical composition as described above (eg, suspended in about 0.01 mL to about 1 mL of a physiologically compatible carrier) and delivered by any suitable means. You may The doses may be repeated daily, weekly, monthly or at other selected intervals as needed or desired. B. Ex Vivo In another aspect, the PEG-modified viruses of the invention are useful for ex vivo transduction of target cells. In general, ex vivo therapy involves the removal of a population of cells containing target cells, the in vitro transduction of the cells, and the subsequent reinfusion of the transduced cells into a human or livestock patient. Such ex vivo transduction is used, for example, in the treatment of cancer when the target cells are dendritic cells or macrophages, and / or when the transgene or other molecule being delivered is highly toxic. It is especially desirable in the case of several genes. However, one of skill in the art will readily select the ex vivo therapy of the invention taking into account factors such as the type of target cell to be delivered, the molecule to be delivered, the condition being treated, the condition of the patient, etc. can do.

Generally, when used in ex vivo therapy, the targeted host cells are treated with 10 ⁵ to 10 ¹⁰ viral particles for each 10 ¹ to 10 ¹⁰ cells in the population of target cells. To infect. However, other suitable levels of ex vivo administration can be readily selected by one of ordinary skill in the art. C. Re-Administration In one aspect, the invention uses a PEG-modified vector of the invention, wherein the MPEG activating group is different from the activating group utilized on the first-administered PEG-modified vector. Provided is a method of delivering a transgene via a PEG-modified viral vector, wherein re-administration or repeated delivery is performed. Therefore, the present invention provides a first activated MPEG (eg, Tresyl-
Delivering a PEG-modified viral vector conjugated with (MPEG)
And may require a second delivery using a PEG-modified viral vector in which MPEG is conjugated with a second activated MPEG group (eg, succinimidyl succinate MPEG, SSPEG). Alternatively, the PEG-modified viral vectors of the present invention may be used in a dosing regimen that utilizes non-pegylated viral vectors. Thus, the PEG-modified viral vector of the invention may be delivered prior to delivery of the non-pegylated viral vector. In another example, delivery of the PEG-modified viral vector of the invention may be followed by pre-administration with a non-pegylated viral vector.

The following examples are provided to illustrate methods of making the compositions of the invention and methods of the invention. These examples do not limit the scope of the invention. Those skilled in the art will recognize that certain reagents and conditions, although outlined in the examples below, can be modified which is meant to be covered by the spirit and scope of the invention. Let's do it. Example 1-Preparation of linked adeno-associated virus (AAV) vectors Preparation of Recombinant AAV (rAAV) rAAV containing LacZ was prepared by the published method. RAAV, AAVCMVLacZ, used in the following studies, is a human cytomegalovirus (
It contains the AAV 5'and 3'ITRs flanking the E. coli β-galactosidase reporter gene expressed under the control of the CMV) promoter. Another rAAV used in these studies is AAVAlbα1AT, which contains the AAV 5 ′ and 3′ITR flanked by the human α1-antitrypsin (α1AT) gene expressed under the control of the albumin promoter. These are described in [G. -P. Gao et al . , Hum. Gene Ther. , 9 :
2353-2362 (1998)], using the B50 cell line.

B50 cells (a HeLa-based cell line expressing AAV Rep / Cap proteins) were treated with wild type Ad5 for 24 hours at a multiplicity of infection of 10 and then at the same multiplicity of infection (MOI) for the Ad-AAV hybrid. Vector [K. J. Fish
er et al . Virol , 70 : 520-532 (1996)] for an additional 48 hours. At this point the cells are harvested, resuspended in 10 mM Tris buffer pH 8.1, and frozen / dried 3 times in a dry ice / ethanol and 37 ° C. bath.
It melted according to the melting cycle. Benzonase (Nucomed Pharma (Nuco
MedPharma), pure grade) was added to the mixture (50 U / ml, final concentration) and the lysate was incubated at 37 ° C. for 30 minutes. The lysate was clarified by centrifugation at 3700 g for 20 minutes and the supernatant was collected. The established method [S. Zolotukin et al., Gene Ther. , 6 : 973-
985 (1999)], and the lysate was added to 20 mM PBS (pH 7.5).
), POROS HE 20 equilibrated with 250 mM NaCl (Perspective Biosystems).
) The column was loaded. After the lysate entered the resin, the column was washed with 10 column volumes of equilibration buffer. The vector was added to 40 mM 20 mM PBS (pH 5.5) 40
Elution from the column with 0 mM NaCl directly onto a POROS 50 PI column. Fractions from the second column were collected, concentrated, and incubated at 56 ° C for 10 minutes to inactivate any residual adenovirus in the preparation. B. Viral vector conjugation A monomethoxy derivative of polyethylene glycol (approximately MW 5000)
Selected for binding to rAAV particles. This compound must first be chemically activated before the coupling reaction. Three types of activated monomethoxypolyethylene glycol (MPEG): tresyl-MPEG (TMPEG), succinimidyl succinate MPEG (SS-PEG) and cyanuric chloride MPEG.
(CC-MPEG) was used in this study. All activated PEGs are sigma
Obtained from Sigma Chemicals (St. Louis, MO). In all cases, the method the binding reaction, which is established [Delgado, Bio Technol. Appl. Biochem. , 12 : 119-128 (199
0); Kita, Drug Design & Delivery , 6 : 157.
-167 (1990); Jackson, Analytical Biochem m. , 165 : 114-127 (1987)]. TM
For conjugation with PEG, column purified AAV was dialyzed in 10 mM potassium phosphate buffer pH 7.4. The viral bands were labeled with SS-PEG and C.
For the conjugation reaction with C-PEG, 0.2M sodium phosphate (pH 7) was used.
． 2) and 0.1 M sodium tetraborate (pH 9.2) buffer.
We found that 10: 1 (300 μg / 3 mg) for conjugation with each type of PEG.
It was found that the PEG: virus ratio of () (amt PEG: amt AAV protein) provided highly efficient reaction times and resulted in minimal loss of viral infectivity. AAV mixed with a polymer unable to covalently attach to the virus, MPEG not activated, served as a control for all in vitro and in vivo transduction experiments. All coupling reactions were performed at room temperature with gentle agitation. The reaction was stopped by the addition of a 10-fold excess (relative to PEG concentration) of L-lysine. Unreacted PEG, excess lysine, and reaction by-products cause the preparation to
Elimination was by passing through a Sephadex G-50 column equilibrated with 10 mM potassium buffered saline (KPBS) pH 7.4. Fractions containing virus were identified by UV spectrophotometric analysis at 260 nm and combined for further study. Example 2-Infectivity Assay of Bound and Unbound Viral Aliquots of bound and unbound AAV were serially diluted in DMEM supplemented with 2% FBS and added to 293 cells. Preparations were removed and replaced with complete medium 2 hours post infection. Twenty hours after infection, cells were washed with PBS, fixed with 0.5% glutaraldehyde and washed twice with PBS containing 1 mM MgCl ₂ . β-Galactosidase expression was 1 mg in PBS containing 5 mM K ₃ Fe (CN) ₆ , 5 mM K ₄ Fe (CN) ₆ 3H ₂ O and 1 mM MgCl ₂ for 2 hours at 37 ° C. in the dark. / Ml substrate, 5-bromo-4
It was measured by incubation with chloro-3-indolyl-β-galactoside (X-gal). Lac ⁺ cells were coated from a minimum of 20 microscope fields. Percent infectivity was calculated as L at various time points relative to the number of Lac ⁺ cells at the start of the test.
It was determined by calculating the ratio of the number of ac ⁺ cells.

Unbound AAV showed the most significant loss of titer (29
%)experienced. The CCPEG vector was the least stable of all PEGylated preparations with a loss similar to that of the native virus. Simple addition of PEG to the preparation did not compromise virus titer.
This is because the addition of non-activated MPEG to the virus preparation did not significantly affect infectivity. Upon further study, it was found that the loss of infectivity of the CCPEG vector could be attributed to the formation of large aggregates due to the cross-linking of multiple virions. However, for a shorter period of time, CCPEG the vector
Reacting with lesser aggregates and transduction efficiency and stability is greatly enhanced even if less than 100% modification is achieved. Example 3-Fluorescamine Assay The fluorescamine assay was used to estimate the extent of modification of the AAV capsid by activated polyethylene glycol. The assay is an established method [Stocks, Analytical Biochem. , 154 : 232-
234 (1986)]. Samples were taken 30, 60, 90, 120 and 480 minutes after the start of the binding reaction. Make serial dilutions from 1.
Made from each sample in a volume of 5 mL of 10 mM sodium phosphate buffer pH 7.4. Fluorescamine (0.3 mg / ml, in acetone (0.5 ml)
Sigma Chemicals, St. Louis, MO) was added to each dilution with vortexing. The fluorescence of the sample
A spectrofluorometer with an excitation wavelength of 390 nm and an emission of 475 nm (Photon Technology Int.
international), Mammoth Junction, NJ). The resulting fluorescence is proportional to the concentration of free amino groups on the viral capsid. A standard curve was generated for each time point by plotting protein concentration versus fluorescence units. The extent of modification was obtained as the ratio between the slopes of bound and unbound virus at similar time points.

[0047]

[Table 1]

The reaction with CCPEG was complete after 60 minutes (when the titer had dropped by less than 10% and few aggregates had formed). The transduction efficiency of AAV was enhanced by its association with TMPEG over the entire reaction period. The reaction was completed in 30 minutes. Binding of AAV to SSPEG was completed within 15 minutes with minimal loss of titer over the entire reaction period. Example 4-Characterization of PEGylated AAV preparations Several physical tests were performed to confirm that activated PEG molecules were successfully attached to the AAV capsid. A. Partition Assay The partition coefficients of native and PEGylated virus have been previously described [Del.
gado, J .; Biochem & Biophys. Meth. , 29:23
7-250 (1994)]. Partition assay is 0.15M Na
4.75 in 0.01 M sodium phosphate buffer containing Cl, pH 6.8
% (W / w) PEG8000 (Sigma Chemicals
Als), St. Louis, Mo.) and 4.75% Dextran T500 (
Amersham Pharmacia Biotech (Amersham Pharma)
Cia Biotech), Piscataway, NJ) 1g double layer system
Was carried out at 25 ° C. in a single microcentrifuge tube containing The bilayer system is 40% PEG,
20% dextran, 0.44M sodium phosphate buffer, pH 6.8 and 0
． Prepared from a 6M sodium chloride stock solution. AAV and PEGylated AAV were incorporated into the system by replacing 0.1 g of water used to prepare the phases with 0.1 g of virus in binding buffer. The samples were mixed by inversion 30-40 times and left to stand under gravity until complete separation of layers was achieved. Aliquots from the top and bottom layers were taken from Bio-Rad (Bio-Ra).
d) Protein assay analyzed for protein concentration by microplate assay using bovine serum albumin as reagent and standard. Partition coefficient (K)
Is determined by the ratio between the protein concentrations in the upper and lower layers.

[0049]

[Table 2]

Partitioning of viral preparations in an aqueous bilayer system and calculation of partition coefficient (K) is
It was demonstrated that EGylation significantly modified the viral capsid. K values moved from 0.68 for unlabeled virus to 1.26 and 1.01 for CCPEG and SSPEG preparations, respectively. TMPEG preparations are 1.
The highest level of binding was established with a K value of 37. B. Zeta potential The zeta electron (electrophoretic mobility) of each PEGylated virus preparation was determined to be 10
Laser Doppler anemometry (Zetasizer) in a thermostatted microelectrophoresis cell as previously described with a 25-fold dilution in mM potassium phosphate buffer pH 7.4.
) 3000, Malvern Instruments
(Ments), Southborough, Mass.). Each reported value is the average of 3 separate measurements.

The surface charge of AAV capsids was effectively altered by the PEGylation process. This is because the zeta potential level changed from -9.2 mV for the native vector to -6.4 mV and -5.9 mV when coupled to TMPEG and SSPEG, respectively. The CCPEG preparation demonstrated maximal migration towards neutrality up to -5.1 mV. C. Electron Microscopy An aliquot of bound virus particles was added to 1% glutaraldehyde, 0.05
It was fixed in M cacodylate buffer pH 7.4 at room temperature for 10 minutes. The solution was spread on a 200 mesh carbon coated grid (Electron Microscopy Sciences, Fort Washington, Philadelphia). After 10 minutes, the excess suspension was filtered through a grid with filter paper. The grid was stained with 2% uranyl acetate for 1 minute, rinsed and dried. Viral particles were viewed using a Philips CM-10 transmission electron microscope (Eindhoven, The Netherlands).

The average particle size of PEGylated AAV in three separate animal studies is provided in the table below.

[0053]

[Table 3]

Example 5-Neutralization Assay The PEG modification method of the present invention (ie, "pegylation").
To determine whether it was able to preserve AAV transduction efficiency in the presence of neutralizing antibody (NAB), the bound AAV preparation was treated with multiple doses of AAV (1 x 1).
0 ¹² particles) were incubated in the presence of sera from C57BL / 6 mice that had already been given or serum from non-immunized C57BL / 6 mice.

A 200 microliter aliquot (equivalent to a multiplicity of infection of 50) was applied in triplicate to 84-31 cells (293 based E1 / E4 complementing cell line). Two
The vector was replaced with complete medium after a period of incubation. 48 hours post infection cells were washed with PBS and fixed in 0.5% glutaraldehyde for 10 minutes,
Then, the plate was washed twice with PBS containing 1 mM MgCl ₂ . Transduction levels are reported as the ratio of cells transduced by virus incubated with immune serum to cells transduced by virus incubated with non-immune serum.

The transduction efficiency of the PEGylated vector was not affected by the presence of 100- and 1000-fold diluted immune sera, whereas the unbound preparation was reduced by 49% and 32% respectively. . At the highest dilution, the transduction efficiency of the PEGylated vector dropped by approximately 30%, but was significantly higher than that of the unbound virus (Student's t-sample, p ≦ 0.01).
).

Mouse sera were incubated at 56 ° C. for 30 minutes to inactivate complement, followed by dilution with DMEM in a 2-fold increase starting with a 20-fold dilution.
NAB was analyzed. Mix each dilution (100 μl) with virus, 37 ° C
Incubated for 1 hour at and applied to 84-31 cells in 96-well plates (2 × 10 ⁴ cells per well). After 1 hour at 37 ° C., 100 μl of DMEM supplemented with 20% FBS was added to each well. The cells were incubated for 24 hours and the expression of green fluorescent protein (GFP) was FluoroImaging (Molecular Dynamics).
The measurement is made by means of ulal Dynamics)). NAB titer of 50 cells
Calculate as the highest dilution in which% stains green.

Anti-AAV NAB was reduced by 20% and 50% respectively after administration of the vector conjugated to TMPEG and SSPEG. The CCPEG preparation is the least efficient vector for this application, and it is a transgene.
The levels of ne) fell 10-fold below the other vectors at all time points and to undetectable levels 60 days after injection. NAB levels were only mildly reduced by this preparation. Example 6-Administration of PEG-Modified Vectors in Immunocompetent Animals The following studies show that PEGylation of the invention affects the transduction efficiency of AAV when administered to the muscle or liver of immunocompetent animals. Prove that it doesn't.

C57BL / 6 (H-2 ^b ) mice (6-8 weeks old) were tested by Jackson Laboratories (Bar Harbor, Maine).
Purchased from. To determine the effect that PEGylation has on AAV transduction, unbound native AAValbα1AT or AAVCMVlacZ (the same virus as described above, combined with various polyethylene glycols) and unactivated MPEG were mixed. Virus in the muscle (50 μl PB
5 × 10 ¹⁰ particles in S) or in the liver (1 × 10 ¹¹ particles in 100 μl PBS). The animals were necropsied 14 days post-injection and the tissues were split in half and the OTC compound (Miles D.
ignostics), Echhardt, Indiana)
rapid immersion in el-away mold and frozen sectioning (cryo).
Sections) were snap frozen in a dry ice / isopentane bath. The remaining tissue was placed in cold DMEM and processed for expression assay as described below. A. β-gal Assay Treated tissues are excised from freshly euthanized animals and washed twice in cold PBS. When processing a large number of samples, the excised tissue should be cold DMEM.
Store in less than 2 hours. Tissue is treated with 4 mM Pefabloc
ock) (Boehringer-Mannhei
m)), 1 mM phenylmethylthultonyl fluoride (PMSF), 1 mM benzamidine, 1 μg / ml pepstatin, 5 μg / ml aprotinin (Sigma (Si
gma)), 1 μg / ml leupeptin, 0.5 mM EDTA and 0.5 m
Lysis buffer containing M dithiothreitol (DTT) (β-gal ELIS
A kit, Boehringer-Mannheim
Supplied with m)) Rinse in. The organization is called Brinkman.
n) Homogenize in 1 ml lysis buffer using Polytron. After homogenization, the extract is centrifuged at 14,000 rpm for 10 minutes. The protein concentration of the clarified supernatant was measured by a microplate assay using Bio-Rad DC protein assay reagent and bovine serum albumin as standard. The extracts were snap frozen in a dry ice / ethanol bath and stored at -80 ° C until assayed. The β-gal concentration was determined by the β-gal ELISA kit (Boehringer-Mannheim (Boehr) according to the manufacturer's instructions.
(Inner-Mannheim)) is used for the measurement by enzyme-linked immunosorbent assay (ELISA).

After intramuscular injection of AAVCMVlacZ vector, the initial transduction efficiency of TMPEG, SSPEG and unconjugated virions was not statistically significant (Stud.
ent's t-sample, p ≦ 0.05); in each case, the transgene
The expression of ne) was generally stable for the duration of the experiment. The CCPEG preparation was the least efficient and produced transgene levels that were 10-fold lower than the other vectors at each time point, with gene expression dropping to undetectable levels 60 days after injection. did. All animals receiving intramuscular injections of either native or conjugated preparations developed high levels of neutralizing antibodies to the AAV capsid protein. B. Human α-1 Antitrypsin Assay The concentration of human α1AT in mouse serum has been previously described [W. D. Xiao et al . Virol. , 73 : 3994-4003 (1999)], and measured by enzyme-linked immunosorbent assay (ELISA). Microtiter plates (MaxiSorp, Nunc) were coated with 100 μl of rabbit anti-human α1AT antibody (100 ×, Sigma) at 4 ° C. overnight, blocked, and 4 with standard or sample. Incubated overnight at ° C. After washing, primary antibody (1000 times, goat anti-human α1AT, Sigma (Sig)
ma)) was incubated with the captured antigen for 2 hours at room temperature. Secondary antibody (anti-goat IgG-peroxidase conjugate, Sigma, 10,0
00 times) was added at room temperature for 2 hours. ABTS reagent (Boehringer Mannheim (
Boehringer Mannheim)) and add a concentration of 405n
Determined from the absorbance reading at m. Assay sensitivity is 0.3 to 30 ng /
ml.

[0061]   When animals were injected intravenously with the AAValbα1AT vector, TMPEG and
And SSPEG preparations only produce the original vector at the rate at which gene expression reached a steady state.
It was different from Tar. 1 x 10^FourPeak of gene expression equal to ng / ml α1AT
Original vector as well as SSP when levels are assessed 14 days post injection
Shown for EG and TMPEG combined vectors. Example 7-AAV-specific immunoglobulin analysis   Serum samples from mice have been previously described [N. Chirmul et al.J ． Immunol. ,163: 448-455 (1999)]
AAV-specific isotype-specific immunoglobulin (IgG1, IgG
2a, IgG2b, IgG3, IgM). Micro titer
Rate (MaxiSorp, Nunc) 0.1
100 μl of AAV antigen in bicarbonate buffer, pH 9.6 (5 x 10 / ml) ^Ten Individual particles) at 4 ° C. overnight and 0.05% Tween 20
Wash 4 times with PBS containing and 3 at room temperature in PBS containing 3% BSA.
Blocked for hours. A 100-fold diluted serum sample was added to the antigen-coated plate.
And incubated overnight at 4 ° C. Plate, 0.05%
Wash 4 times with PBS containing Tween 20, and 100
0-fold diluted biotin-conjugated rat anti-mouse anti-IgG1, IgG2a, IgG2b,
IgG3, IgM (PharMingen, CA
(San Diego, State) for 3 hours at room temperature. With the plate up
Wash with a cage and then use alkaline phosphatase-conjugated avidin (Sigma Chemical   Company (Sigma Chemical Company), Missouri
A 10,000 dilution of St. Louis) was added for 2 hours at room temperature. After four washes
, P-nitrophenyl phosphate in diethanolamine buffer was added (Sigma
Chemical Company, Sigma
St. Louis, Z.) and optical density on a microplate reader (da
Inatec Laboratories (Dynatech Laboratories)
, Chantilly, Virginia) at 405 nm.

Characterization of anti-AAV antibody isotypes was performed by using the CCPEG preparation as an IgG1 antibody (
This was the case, as it was the only formulation that significantly increased production of the Th2 pathway). Example 8-Immunization Experiments Results from immunization studies using rAAV pegylated according to the present invention include:
It demonstrates that the pegylated rAAV of the invention allows for enhanced gene expression. A. PEGylation allows for significant gene expression in immunocompetent animals after pre-exposure to the native vector. As shown herein, the transduction efficiency of several PEGylated vectors is relative to the native vector in vitro. Not affected by the presence of neutralizing antibody (NAB). To determine if this effect could also occur in vivo, animals were placed 30 days after the initial immunization with the same dose of native AAV.
Challenged with a second dose of PEGylated vector (1 × 10 ¹¹ particles iv or 5 × 10 ¹⁰ particles im). The second vector is AAVCMVlacZ for intramuscular injection and AAValbα1AT for intravenous injection.
Met. Animals that received two doses of the original vector intramuscularly did not demonstrate significant levels of gene expression upon rechallenge. Although animals receiving the TMPEG preparation had significant levels of gene transduction upon re-administration, it was approximately 7.5-fold reduced compared to animals that received no treatment. The level of gene expression obtained with the SSPEG preparation was substantial, but less than 10-fold lower than untreated animals. Significant levels of gene expression were also found in the CCPEG preparation, but this was the least effective.

Administration of two doses of native AAV failed to produce a significant level of gene expression according to the second vector intravenously. All PEG delivered intravenously
The modified preparation produced high levels of gene transfer after intravenous immunization with the native vector, which is similar to that observed in naive animals,
It ranged from 100% of never received TMPEG to 40% of the original CCPEG. B. PEGylation allows for significant gene expression by unbound AAV in immunocompetent animals after pre-exposure to PEGylated virus. Effect of humoral immune response to PEGylated vector on gene transfer with native virus. Researched. Mice immunized with various preparations of PEGylated AAV were given a second intramuscular injection of 5 × 10 ¹⁰ particles of the original vector. Significant levels of gene expression, despite the presence of anti-AAV antibodies,
Detected in animals immunized with PEG and SSPEG preparations. Mice immunized with the TMPEG preparation produced the most significant level of gene expression upon rechallenge with native virus, but still somewhat to some extent than that seen in naive animals. it was under.

The liver-directed native virus produced significant levels of gene expression in animals immunized intravenously with PEGylated AAV. Mice immunized with SSPEG and CCPEG preparations demonstrated significant levels of gene expression upon re-dosing as measured by α1AT serum levels, which were administered to naive animals. 42 than that seen with single dose vector
% (SSPEG) and 21% (CCPEG) only. The native vector caused the most significant level of gene expression in the animals that were immunized with TMPEG despite the fact that they had the highest levels of NAB to the native vector prior to administration. C. Repeated administration of PEGylated AAV produces significant levels of gene expression in liver but not muscle C57BL / 6 receives two consecutive intramuscular administrations of PEGylated AAV did. The ultimate goal of these experiments was to evaluate the gene transfer efficiency with a particular form of PEGylated AAV after a previous immunization with the same type of PEGylated vector. Re-administration of the same type of PEGylated vector was substantially reduced when compared to intramuscular injection into naive animals, with each PEG
For the modified vector, 20-fold reduction with CCPEG to 20-fold with SSPEG
The range reached to 0 times decrease. All animals developed significant titers of anti-AAV NAB. When administered intravenously, gene expression after 2 doses of the CCPEG preparation resulted in α1AT serum levels comparable to those seen in naive animals, despite the presence of anti-AAV NAB. Let The SSPEG preparation produced a slightly reduced gene expression after re-dosing (ie 50% of naive animals). Transduction of TMPEG preparations upon re-administration surpassed that of animals that had never received treatment. Example 9-Preparation of Bound Adenovirus Vectors An E1 / E3 deleted adenovirus vector (H5.010.CMV.LacZ) expressing β-galactosidase was used in these studies and was established. A variant of the method was used to amplify in 293 cells and purified from cell lysates by double banding on a CsCl gradient (Graham and Van.
der, Virology , 52 : 456-467 (1973)). Aliquots of virus were desalted on Econo-Pac 10DG disposable chromatography columns (Bio-Rad, Hercules, Calif.) And equilibrated with the respective buffers for optimal binding. (See below). Virus concentration was measured by UV spectrophotometric analysis at 260 nm. Transduction titer (ie LacZ forming unit or lfu)
Was determined by limiting dilution infection of 293 cells. The particle / lfu ratio of both bound and unbound virus was approximately 100. The protein content of Ad preparations was measured by a microplate assay using Bio-Rad DC protein assay reagent and bovine serum albumin as a standard.

These adenovirus vectors were conjugated with three types of activated PEG as described in Example 1B. Example 10-Binding of activated MPEG to adenovirus capsids occurs rapidly with minimal loss of viral infectivity A TMPEG-adenovirus (Ad) preparation retains viral titer throughout the reaction period. Whereas, the SSPEG preparation lost 70% of its original titer when the conjugation reaction was complete. Immature termination of the SSPEG reaction (ie it is 70%
75 minutes completed) resulted in a preparation with 90% residual activity. CCP
Modification of the virus in EG was completed in 90 minutes, however, the original infectious 1
Only 1% remained. Fractionation of this preparation on a Sephadex G50 column gave rise to distinct virus populations. The first peak represents a highly infectious PEGylated monomeric virus, while the second peak is an aggregated virus with low activity (by electron microscopy and static laser light scattering, data are (Not shown). Only the first peak was isolated and used in additional studies. Addition of non-activated MPEG to the Ad preparation had no effect on infectivity compared to the vector in buffer alone (data not shown).

The significant loss of titer with CCPEG can be attributed to the production of large viral aggregates by extensive cross-linking of the polymer with the viral capsid. It is manufactured as a mixture of two isoforms, one that can bind to a protein by a single amide bond and another that can form two amide bonds with a target protein. To be done. Once the aggregates were removed, the remaining viral suspension was found to be highly infectious and extremely stable under a variety of storage conditions. S
The SPEG preparation also experienced a significant drop in potency after 1 hour binding at room temperature.
No aggregation phenomenon was detected with this compound, however, loss of potency could be attributed to the binding of multiple SSPEG molecules to a single lysine residue. 7 of the available lysine residues were used to maintain substantial viral infectivity with significant modification.
A reaction time of 75 minutes was selected in which 0% was modified and the titer was maintained. While this was the only protocol that did not completely modify the viral capsid, the resulting vector efficiently escaped neutralization by immune sera and was PEGylated by other preparations under a variety of storage conditions. We were able to maintain the highest titers for things. These data indicate that complete modification of the viral capsid with this polymer is not a strict requirement for the production of highly efficient viral vectors. Example 11-Ad-PEG Conjugate Characterization Several physical tests were performed to confirm that the activated PEG molecule was successfully bound to the adenovirus capsid. Partition assay and zeta potential were performed as described in Example 4.

Partitioning of viral preparations in an aqueous bilayer system and calculation of partition coefficient (K) is
It was demonstrated that the viral capsid was significantly modified. K values moved from 0.7 for unlabeled virus to 1.76 and 1.96 for TMPEG and SSPEG preparations, respectively. The CCPEG preparation has a K of 3.56
With the value, the highest level of binding was demonstrated.

Zeta potential analysis showed that the surface charge of the adenovirus capsid changed significantly from −48.1 mV to −27.8 and −24.2 mV when coupled to TMPEG and SSPEG, respectively. . The CCPEG preparation is -1
Demonstrated maximum transfer towards neutral up to 6.2 mV. Example 12-Adenovirus-PEG Complex Protected from Neutralization by Immune Serum The PEGylated preparation is added to HeLa cells in the presence of neutralizing antibodies against adenovirus capsid protein and transduced. Levels were compared to those of the same preparation incubated in non-immune serum. This study demonstrates that immune sera were harvested 28 days after intravenous injection of adenovirus vector C57BL / 6.
Performed essentially as described in Example 5 above, except that it was from a mouse.

The transduction efficiency of unbound virus was significantly reduced by the neutralizing antibody. TM
The PEG preparation partially shielded the virus from neutralization, while SSPEG and C
Transduction of CPEG preparations was not affected by the presence of neutralizing antibody. Example 13-Stability of PEGylated Adenovirus As shown in the study below, MPEG was added at 42 ° C and 25 ° C when added at concentrations similar to those used in the PEGylation reaction. It extended the stability of the virus beyond that of the unbound adenovirus, but the virus titer was P
It was substantially lower than that of EGylated virions. Precipitates were finally detected in these preparations, which contributed significantly to the loss of transduction. However, the addition of this excipient to the adenovirus formulation at 4 ° C significantly increased stability over that of the native virus for up to 5 months due to its ability to maintain capsid assembly at this temperature. Raised. The addition of glycerol to the PEGylated preparation is
It did not affect the stability of the virus at -20 ° C. This result is promising because it can eliminate glycerol (and its associated toxicity) from preparations for clinical use. Although each PEGylated preparation lost about 1 log titer over 4 months, they were stored in the absence of any additional cryoprotectant. Addition of carbohydrates, surfactants and other stabilizers can increase stability and reduce loss of potency to negligible levels. A. PEGylated adenovirus is significantly more stable than unbound virus under a variety of storage conditions The ability of PEGylation to stabilize virus over a wide range of temperatures was evaluated. The preparations were stored in potassium buffered saline (KPBS) at −20 ° C. and 4 ° C. with or without the addition of 10% glycerol. Unbound adenovirus underwent a 1 log drop in titer after storage at 4 ° C. for 8 hours, dropping to undetectable levels after 7 days of storage. This rapid degradation of the viral capsid is easily detected by electron microscopy. After 24 hours at 4 ° C., the unbound virus preparation consisted mainly of a single viral capsid protein, mostly hexon; only some intact virus particles could be detected. The CCPEG preparation was the least stable of the remaining preparations. This is because the titer dropped by one log after incubation at 4 ° C. for 24 hours. Electron micrographs show clear evidence that this preparation is significantly more stable than unbound virus, as the photographs taken after 12 days of storage show intact virions. The TMPEG preparation demonstrated a negligible loss of titer at 1 week at 4 ° C; after 42 days the titer dropped by two logs and remained at this level for the duration of the study. At this point, the preparation consisted primarily of intact viral capsid, with only a few impaired virions. The SSPEG preparation underwent an initial drop in titer of 1 log after 4 hours at 4 ° C. and maintained titer for up to 150 days. This preparation was indistinguishable from freshly purified virions upon examination by electron microscopy after 120 days storage at 4 ° C.

Stability studies of the PEGylated preparation at -20 ° C showed that the addition of glycerol was not necessary to maintain the titer. In the absence of glycerol, the titer of unbound virions dropped 5.5 logs in KPBS after 15 days at -20 ° C.
Viral degradation continued constantly for the remainder of the study at a rate of 1 log per month. Addition of 10% glycerol significantly increased stability. This is because only one log unit of the titer was lost in the unbound preparation stored at -20 ° C for 4 months. SSPEG-conjugated virions were the most stable of all the preparations tested, with a drop in titer of 0.8 log at −20 ° C. over 120 days; addition of glycerol slightly stabilized the stability. Only raised. The titer of the TMPEG preparation was most sensitive to the addition of glycerol. The glycerol-free preparation demonstrated a 1 log drop in storage after 90 days, while the titer of the glycerol-containing preparation dropped only 0.5 log units. CCPEG virion is
Least stable with a 4 log reduction of infectious virus after 90 days storage at -20 ° C; glycerol did not help. B. PEGylation Enhances Adenovirus Stability Under Extreme Storage Conditions Even though the stability data for PEGylated virus preparations at 4 ° C and -20 ° C were very promising, these conditions were It may also require shipping on ice to various clinical locations. Samples were stored at 25 ° C. and 42 ° C. in KPBS to assess the potential delivery of the vector under ambient conditions. Upon storage at 25 ° C., unbound adenovirus dropped in titer at a log rate per day. Addition of monomethoxypoly (ethylene) glycol to adenovirus preparations slightly increased the stability of unbound adenovirus preparations with loss of titer at an average rate of 0.6 log units per day. .

The CCPEG preparation was very stable at room temperature. 5.84 × 10 ¹⁰ lfu / m
An initial drop in titer from 1 to 3.18 × 10 ¹⁰ lfu / ml was detected after 6 hours, which was stable for the following week. While the TMPEG preparation was stable for 24 hours, the titer of the SSPEG preparation remained constant for up to 5 days, which would allow for preferential delivery of the vector in the absence of dry ice. When stored at 42 ° C., the titer of unbound adenovirus preparation was initially approximately every 45 minutes.
It descended at a logarithmic steady rate. Addition of inactivated MPEG delayed the loss, which was finally diminished by 2 logs over 3 hours. TMPEG and CCPE
The G preparation demonstrated a similar rate of degradation with a titer of 1.5 log units falling over the duration of the test. The SSPEG preparation was the most stable under these conditions for 18 hours with a negligible loss of titer. The SSPEG preparation demonstrated a negligible loss of titer at 42 ° C for up to 8 hours. Thus, the preparation was able to survive exposure to extreme temperatures during shipping without significant loss of titer. Example 14 - Immune administration of PEG of adenoviral vectors into competent animal ^{C57BL / 6 (H-2 b} ) mice (6-8 weeks old) Jackson Laboratories (Jackson Laboratories) (Bar Harbor, Me)
Purchased from. The preparations were administered either via the tail vein (1 × 10 ¹¹ particles in 100 μl KPBS) or intratracheally (5 × 10 ¹⁰ particles in 50 μl KPBS). Animals were necropsied after 4 days and tissues were excised, washed twice in cold PBS and stored in cold DMEM for processing. Tissues were homogenized in 1 ml lysis buffer using a Brinkman Polytron. The extract was centrifuged at 14,000 rpm for 10 minutes. The protein concentration of the supernatant was measured using the Bio-Rad DC protein assay reagent and bovine serum albumin as standard. Extracts were snap frozen in dry ice and stored at -80 ° C until assayed. β-gal concentration was measured using a β-gal ELISA kit (Boehringer-Mannheim) according to the manufacturer's instructions.

When administered to the lung, unbound virus is 1 per mg protein.
． 42 × 10 ⁴ ± 3.6 × 10 ³ pg of β-galactosidase was produced. CCP
The EG and SSPEG preparations produced 2-fold higher β-galactosidase expression levels than unbound virus. The TMPEG preparation demonstrated the greatest increase in transduction with a 2.5-fold increase in β-galactosidase. Addition of inactivated MPEG to the Ad preparation increased transduction to a level three times higher than that of unbound virus.

Administration of unbound adenovirus resulted in intravenous administration of protein 1m in the liver.
A β-galactosidase level of 3.0 × 10 ⁶ ± 1.1 × 10 ⁵ pg / g was produced. Liver in vivo transduction is 6 with TMPEG, CCPEG and SSPEG
It was twice as high. The addition of MPEG to the virus preparations increased transduction levels slightly above that of unbound virus.

PEGylation of adenovirus vector enhanced transduction efficiency when administered intratracheally or intravenously. This effect was somewhat unexpected. This is because the majority of lysine residues present on the viral capsids are concentrated on the fiber and penton proteins that are required for binding and entry of the virus into target cells [(Adam et al., Acta Microbiological Academia e Scientificia Hungaricae. , 24 : 181-187 (
1977), Bergelson et al., Science , 275 : 1320-13.
23 (1997), Wickham et al., Cell , 73 : 309-319 (19).
93))]. However, the new physical characteristics of PEGylated viruses may contribute to the observed increase in viral transduction. Zeta potential measurements indicate that adenovirus carries a significant negative charge on the capsid. The surface charge of viral vectors can significantly affect the level of transduction in a variety of target tissues (Fasbender et al., J. Biol . Chem . , 272 : 6479-6489 (1997)). Adenovirus transduction has been found to be inhibited by static repulsion between the virus and a negatively charged sialic acid residue on the cell surface (Arcasoy et al. Am. J. Resp. Cell ). & Molec. Biol. , 17 : 422-435 (1997)).
. PEGylation effectively masks the groups responsible for this charge, creating an environment that appears to favor viral nonspecific interactions with cell membranes. Final PE
Particle size determination of glycated preparations is also a suspension of single viral particles, where each method can increase the number of virions contacted with the cell monolayer and consequently increase transduction efficiency. Was generated. The partition coefficient of PEGylated virions indicates that the modified vector has an increased affinity for the hydrophobic environment that appears to allow for an increased ability to partition indiscriminately across cell membranes.
Earlier studies supporting this theory support the mechanism by which the transduction efficiency of PEGylated adenovirus is enhanced. This is because the permeability of the PEGylated vector across the differentiated monolayer is significantly enhanced (data not shown). Example 15-Evaluation of formulations for use in frozen and lyophilized virus preparations Two buffers acceptable for human use, 10 mM sodium phosphate (DPBS).
) And 10 mM potassium phosphate (KPBS) buffered saline at low pH.
Were evaluated for their ability to maintain. A. Effect of Temperature on Various Formulations Materials: USP Sucrose, β-Cyclodextrin, USP D-mannitol, D (+) trehalose, sorbitan monolaurate (Span)
20) and phosphate buffered saline (PBS) were purchased from Sigma (St. Louis, MO). Potassium dihydrogen phosphate (KH ₂ PO ₄ ), dipotassium hydrogen phosphate (K ₂ HPO ₄ ) and United States Pharmacopeia potassium chloride were purchased from JT Baker (Phillipsburg, NJ). Glycerol, USP, was purchased from EM Science (Gibbstown, NJ). Tertiary amine β-cyclodextrin is available from Cerestar USA, Inc.
Hammond, Indiana). Pluronic block copolymer F6
8 was kindly provided by BASF Ltd. (Mount Olive, NJ). Universal pH indicator is Fisher Scientific (Fisher
Purchased from Scientific (Pittsburgh, Philadelphia).

Sample Preparation: All formulations were prepared under GLP conditions and sterilized by filtration through a 0.22 μm filter (Corning). For all studies, 250-1000 μl aliquots were autoclaved to 3 m.
to 1 clear borosilicate glass vial (Wheaton, Millville, NJ). 13mm unbleached in a vial (gray)
Cover with a butyl rubber stopper (Wheaton) and cut (
Sealed with a tear-off aluminum seal (Wheaton). For pH testing, 10 μl of universal pH indicator was added to monitor the pH change during freezing.

PH Test: Eight samples were prepared for each formulation as described. 4 samples are universal pH indicators [M. A. Croyle et al., Pharm. Dev. Te
chnol. 3 (3): 373-383 (1998); Nema and K. E
． Avis, J. Parental Science & Technology
, 47 (2): 76-83 (1993)]. The remaining sample contained no indicator and was used to determine virus titer after freezing. Sample-
Frozen for 12 hours at 20 and -80 ° C. The pH of the frozen product was measured visually and noted for each formulation. 25 samples containing no indicator
Gently warmed to 0 ° C and evaluated for virus efficacy. The results are shown in Table 4 below.

[0077]

[Table 4]

The pH of the sodium phosphate buffered saline is p when it is frozen at -20 ° C.
The pH dropped from H7.4 to 5.0 and at −80 ° C. to pH 4 (see table above). Addition of glycerol to the buffer did not prevent a drop in pH upon freezing. Differential scanning calorimetry demonstrated that this change in pH was associated with precipitation of buffer components during freezing. Add sucrose or trehalose to the buffer at -80
It was not possible to prevent this phenomenon at ℃, but 1M concentration of each sugar was -2.
Dramatic pH changes were prevented at 0 ° C. Potassium phosphate buffer did not demonstrate a significant change in pH as seen with sodium phosphate buffer (DPBS) at -20 ° C, but experienced a similar drop in pH at -80 ° C. did. Addition of cryoprotectant did not significantly affect the final pH of the preparation. However, for each buffer system, it is important to note that the tertiary amine β-cyclodextrin (TMBCD) was able to maintain physiological pH upon freezing. B. Effect of Temperature on Various Formulations Containing Viral Vectors Viruses were formulated in the following manner and frozen at each temperature for 12 hours. Samples were thawed at 25 ° C and virus titers evaluated to determine if changes in pH significantly affected physical stability.

[0079]   The term lac forming unit (lfu) as used throughout the examples below.
The term means limiting dilution, 293 cells [or adenovirus E1 in the case of AAV.
And cell lines expressing E4, 84-31 cells (K. J. Fisher et al.,J. V irol. ,70: 520-532 (1996))] and histochemical staining.
Rabini lac⁺Β-galactosider, as measured by visual identification of cells
Infectious virus particles present in a preparation of virus expressing ze (lacZ)
Define the number of. Samples serially in DMEM supplemented with 2% fetal bovine serum
Diluted to. 1.5 x 10 medium per well^Four1 seeded with individual cells
Removed from the 2-well plate and placed 0.2 ml of the appropriate dilution on the monolayer.
After 2 hours at 37 ° C, 2 ml of complete medium was added to each well and the infection was
Continued for 16 hours at ° C. At this point the medium is removed and the cells previously described.
[M. A. Croyle et al.Pharm. Dev. Technol.,Three(
3): 365-372 (1998)] as to β-galactosidase expression.
Stained. lac⁺A minimum of 20 microscopic cells (approximately 48,000 cells)
I counted from. The lac forming unit has been previously described [M. A. Croyle et al.
,Pharm Dev Technol,Three(3): 373-383 (1998)
)] Was calculated as follows. Assay sensitivity is 10 to 1 x 10¹²at lfu / ml
It was. 1. Preparation of adenovirus   E. coli β-galactosider under control of CMV promoter
The first-generation adenoviruses that express zeb. L. Grah
am and A. J. van der Eb,Virology,52: 456-46
7 (1973)] and amplified in 293 cells. Virus, Cs
Banded twice on Cl gradient, then equilibrated with each respective formulation
Econo-Pack (Econo-Pac) 10DG disposable chromatography
On Rum (Bio-Rad, Hercules, CA)
Purified from cell lysate by desalting with. The virus concentration is 26
Determined by UV spectrophotometric analysis at 0 nm and lac formation assay. All
Experiments were performed with freshly purified adenovirus. 2. Preparation of adeno-associated virus   E. coli β-galactosider under control of CMV promoter
The production of recombinant AAV2 expressing the ze gene produces cis plasmin at a ratio of 1: 1: 2.
(PAAVlacZ containing AAV ITR), trans plasmid (AAV
P5E18 containing W. 2 Rep and AAV1 Cap [W. Xiao et al.J ． Virol. ,73(5): 3994-4003 (1999)]), and
Helper plasmid (pfΔ13, most of the late genes deleted and E2b region
Adenovirus plasmid with a deletion of 8 kb in the region), 60 in a 150 mm dish
Transfection of 293 cells at% confluence was required.
Transfection was carried out by the calcium phosphate precipitation method. Transfer
Cells were harvested 96 hours after transfection and were previously described [K. J. Fi
sher et al.Nature Medicine,Three: 306-312 (1997)
)] Was subjected to two CsCl gradient purifications. Viruses should be matched to each prescription
It was desalted by dialysis. 3. result   Adenovirus preparations are extremely sensitive to pH changes during freezing.
(Fig. 1A). Preparations maintained at the original pH (7.4) have a titer on melting.
Did not experience a significant decline. Preparations that have fallen by 3 pH units have a pair of titers
Suffered a loss of numbers. Adeno-associated virus (AAV) changes pH during freezing
It was not as susceptible to adenovirus (Fig. 1B). On freezing
AAV preparations that experienced a 3 pH unit drop lost 0.5 log of infectious virus
Lost As mentioned above, potassium phosphate buffered saline is at -20 ° C.
P to a greater extent than sodium phosphate buffered saline when frozen in
H was maintained. Adenovirus stored in 10% glycerol in this buffer system
Has demonstrated superior stability over sodium phosphate buffered preparations. As a result
Therefore, potassium phosphate buffered saline was selected for all formulations unless otherwise indicated.
It was the buffer of choice. Example 16-Stability of Adenovirus Formulations at -20 ° C and 4 ° C   Screening potential formulations and buffer systems for their ability to maintain pH at low temperatures.
After the preparation, a large-scale preparation of the formulated virus was given for a period of 2 years, -20 and
And stored at 4 ° C. Viral titers were measured by lac formation assay before storage.
(T = 0). To evaluate the real-time stability of the vector in each formulation,
4 to 6 vials from the gut and evaluated for transduction efficiency
. The virus titer of each formulation was measured in the same lot stored at -20 ° C in KPBS.
Compared to the titer from the virus produced. Expiration date is a standard practice in the pharmaceutical industry.
Tsutomu [Carstensen, J. T. , Drug Stability: Pri
nciples and Practices. Second edition,Drugs and the Pharmaceutical Sciences. Vol. 68.1
995 New York, NY: Marcel Decker, Inc. (Marc
el Dekker, Inc. ); International Conference on Reconciliation (Internationala)
l Conference on Harmonization (ICH), Harmonized Tripartite Guideline, Stab ility Testing of New Drug Substances and Products , 1993; US Food and Drug Administration Drug and Biologics Sen
Center for Drugs and Biologicals FD
A), Guidelines (1987):Guideline for Submit toning Documentation for the Stability of Human Drugs and Biologics , 1987:
Rockville, Leland], the titers fell to 90% of their original values.
Each preparation was split when it fell (ie, suffered 10% loss (0.1 log)).
Hit

[0080]

[Table 5]

The glycerol preparation was the least stable at -20 ° C. The sodium phosphate buffer preparation lost 10% of its original titer in 2 days (Table 5). Potassium phosphate buffer extended the shelf life of the virus by 12 days. When glycerol was removed and sucrose was added to the preparation, titers did not drop for approximately 1 month. Addition of cyclodextrin to the formulation extended the shelf life to 360 (β-cyclodextrin) and 690 (tertiary amine β-cyclodextrin) days, which was −80 ° C.
Significantly longer than that of the virus in glycerol at ~ 37 days (observation not reported).

[0082]

[Table 6]

All formulations stored at 4 ° C significantly increased virus stability (Table 2). β
-The sucrose formulation containing cyclodextrin and surfactant did not show a significant drop in titer for approximately 1 month. Example 17-Stability of AAV Formulations Initial stability studies with adeno-associated virus have demonstrated that this virus is significantly more stable than adenovirus.

[0084]

[Table 7]

Preparations stored at 4 ° C. in sodium phosphate buffered saline took approximately 4 months to lose 0.1 log of infectious virus (Table 7). 2 similar results
Seen at 5 ° C. The formulation consisting of sucrose, mannitol, Span 20 and protamine extended the stability of AAV to 5 months at 4 and 25 ° C. Similar expiration dates were also shown for this formulation at −20 and −80 ° C. Example 18-Effect of formulation on transduction efficiency in vivo Since adenovirus vectors are suitable for many gene therapy applications, we have investigated the transduction efficiency after intramuscular, intravenous and intratracheal injection in C57BL / 6 mice. The effect of these formulations on the was studied. The transduction efficiency of the prescribed virus,
Compared to phosphate buffered saline and vector in a standard 2% glycerol preparation commonly used in preclinical studies of viral vectors [F. Bore
llini and J. M. Ostrove, "The Transfer of T
technology from the Laboratory to the
Clinic: In Process Controls and Fina
l Product Testing ", Gene Therapy Tech nologies, Applications and Regulation s, A.Meager, editor, 1999, John Wiley & Sons (
John Wiley & Sons), West Sussex, UK, p. 35
9-373]. The 5% glycerol formulation was also included thereby as a semi-toxic control for comparing our formulations. A. In Vivo Testing of Formulated Virus C57BL / 6 mice (6-8 weeks old) were tested by Jackson Laboratories (Jac
Purchased from Kson Laboratories (Bar Harbor, Maine). The preparations were administered via the tail vein (1 × 10 ¹¹ particles in 100 μl formulation), intratracheally (5 × 10 ¹⁰ particles in 50 μl) or intramuscularly (5 × 10 ¹⁰ particles in 50 μl). Was administered at any of the following. Four animals from each group were necropsied either 4 days (intravenous and intratracheal) or 7 days (intramuscular) post injection. Tissues were homogenized in 1 ml lysis buffer using a Brinkman Polytron. The extract was centrifuged at 14,000 rpm for 10 minutes. Protein concentration in the supernatant was measured using Bio-Rad DC protein assay reagent and bovine serum albumin as standard. Extracts were snap frozen in dry ice and stored at -80 ° C until assayed. β-g
The al concentration was measured using a β-gal ELISA kit (Boehringer-Mannheim) according to the manufacturer's instructions. Blood samples were collected from the surviving animals of each group via the retro-orbital sinus 4 and 7 days post injection for evaluation of liver function by an external contract laboratory (Antech, NY, NY). . All animals have a basic LF
Blood was collected before the start of the study for T evaluation. B. Results A significant increase in transgene expression was detected after intramuscular injection of sucrose / β-cyclodextrin and 5% glycerol formulations. Other formulations did not exert gene expression in muscle and were well tolerated. Whereas the sucrose / tertiary amine β-cyclodextrin formulation increased viral transduction efficiency in the lung, transduction efficiency was reduced by the 5% glycerol preparation. Signs of acute inflammation were detected in lungs from mice fed this preparation (data not shown). This effect was not detected in animals given the other formulations. All formulations tested slightly enhanced transduction efficiency when administered intravenously. 5%
Glycerol preparations exceed SGs routinely observed after administration of vector.
It caused a significant increase in OT / SGTP levels. This was not seen with the other formulations. Example 19-Development of Lyophilized Viral Vectors Even though the formulation efforts described herein significantly increased the stability of adenovirus at -20 and 4 ° C, they were still shipped on ice. It seems to need. Freeze-drying, a technique commonly used to extend the chemical and physical stability of labile compounds at ambient temperature, was considered as a practical alternative. The freeze-drying process consists of three stages: freezing, primary drying and secondary drying. During the freezing step, the samples are frozen in the range -40 to -50 ° C. When the product is completely frozen, the pressure in the lyophilization chamber is reduced and heat is applied to the product. Under these conditions, water is removed by sublimation (phase change from solid state to direct vapor state without the appearance of an intermediate liquid phase). As freeze-drying proceeds, the thickness of the frozen layer decreases. This is the primary dry period. After primary drying, additional drying is needed to remove any water remaining in the product. During secondary drying, heat is slowly added to the maximum acceptable temperature to maintain product viability and maintained at this level until the process is complete.

Large batches of adenovirus were purified and divided into 5 separate groups.
Each group was desalted into various formulations. Excipients are Timasheff, Carpent
er and others [Carpenter, J. F. And J. H. Crowe, Cry obiology , 25 : 244-255 (1988); Crowe, J. et al. H.
Et al., Cryobiology , 27 : 219-231 (1990); Carpe.
inter, J. F. And J. H. Crowe, Biochemistry , 28 :
3916-3922 (1989); Timasheff, S .; N. , Annu Rev Biophys Biomol Struct. , 22 : 67-97 (
1993]], in order to maintain the protein conformation during the freeze and desiccation steps, we chose according to their ability to displace water and form a protective shell around the viral capsid. The initial titer of the lot was 1 × 10 ¹¹ lfu / ml. Aliquots of 1 ml were placed in glass vials and under standard protocol (pressure of 30 mtorr, -40 ° C for 2 hours, -33 ° C for 12 hours, -25 ° C for 5 hours, 10 ° C for 4 hours and 20 ° C for 8 hours). Lyophilized.

In each lyophilization study, multiple viruses were split into two groups, one half desalted to the desired formulation and the other desalted in KPBS containing 10% glycerol. The latter preparation was stored at -20 ° C and thus served as a control to assess the ability of lyophilization to enhance virus stability. Virus titer -2
It was evaluated (t = 0) before storage at 0 ° C. and lyophilization. A vial containing the formulated preparation was transferred to a single-shelf laboratory grade freeze dryer (FTS Systems (FT
S Systems), Stoneridge, NY) and 1 ° C
Cooled to -40 ° C at a rate of / min. Three vials of virus-free formulation were fitted with a lead platinum RTD temperature probe (FTS) and placed in the front, center and back of the lyophilizer to monitor the temperature change of the product during the process. After cooling, all samples were lyophilized according to the method specifically made for the particular formulation under study [M. J. Pickal, Freeze-Drying of Pro
tains, Part 1: Process Design. Biopharm . , September 1990: p. 18-27]. After drying was complete, the sample was stoppered under vacuum and cut into a 13 mm aluminum seal (Wheaton (Wh
Eaton)). Samples were stored at 4 ° C until assayed for virus titer. Lyophilized virus was reconstituted with 1 ml of sterile injectable distilled water (USP) and placed in KPBS and 10% glycerol at -20 ° C.
Assayed with samples stored at

The lyophilized preparation in sodium or potassium phosphate buffer alone
It suffered a significant loss of infectious virus of 5 and 4 log respectively (Fig. 2). When sucrose, mannitol and Span 20 were added to the formulation, the titer dropped by approximately 1 log. The titer dropped by less than 1 log in preparations lyophilized in 1M sucrose. Example 20-Effect of virus concentration on glass transition temperature (Tg ') of formulations for lyophilization. Even though all excipients significantly improved the recovery of adenovirus after lyophilization, each preparation was In addition, it suffered a drop in potency attributed to the process itself. Therefore, the formulation was further characterized to maximize virus recovery upon completion of the lyophilization cycle. One parameter that is important to ascertain the required processing conditions is the glass transition temperature (Tg '), the temperature at which the freezing phase in the frozen solution is the maximum freezing concentration at which the residual non-ice phase forms glass. F. Franks, Cryo-Lett ers, 11 : 93-100 (1990)]. When primary drying is performed above Tg ', the freeze concentrate behaves as a viscous liquid, the ice microstructure formed on freezing can be lost, and product recovery is significantly hindered. To be If drying occurs below the Tg ', water can be rapidly removed without disrupting the established microstructure upon freezing, which minimizes product loss during lyophilization [ F. Franks, Development. Biol. Stand. 74 : 9-19 (1991)]. Therefore, the Tg 'of the formulation sets a safe upper temperature limit during primary drying. Since the Tg 'can vary over a huge range (-1 to -50 ° C), determining this value for the proposed formulation is extremely important and the first step in process development.

The Tg ′ of the preparation is very enrichment dependent. That is, buffer salts, amino acids, carbohydrates, and other components added to the formulation can affect Tg '[te Boyy, M. et al. P. W. M. Et al., Pharmaceutical Res . , 9 (1): 109-114 (1992)]. Differential scanning calorimetry (DSC)
Gives a direct measurement of Tg '[Her, H .; M. Et al., Pharmaceutical cal Research , 11 (1): 54-59 (1994); Hatle.
y, R. H. M. , Developments in Biological Standardization , 74 : 105-122 (1990); Hat.
ley, R.A. H. M. a. F. , F. , Journal of Thermal Analysis , 37 : 1905-1914 (1991)].

DSC analysis was performed on a MDSC 2920 (TA Instruments, Newcastle, DE) equipped with liquid nitrogen cooling. The calorimeter was calibrated using indium for temperature and cell constant. Fifty microliters of each formulation was hermetically sealed in an aluminum dish (
Analyzed in TA Instruments.
The cooling rate was 1 ° C / min. The temperature range was -60 to + 25 ° C. The glass transition value (Tg ') is reported as the midpoint of the observed transition. The experiment was performed in duplicate.

Sucrose (1M) in potassium phosphate buffered saline has a glass transition of −34.5 ° C. (see Table 8 below).

[0092]

[Table 8]

When adenovirus was added at a concentration of 1 × 10 ¹¹ particles per ml, T
g ′ rose to −33.9 ° C. Addition of another log of virus pushed the glass transition temperature to -32.0 ° C. This effect is universally seen in proteins [M.
J. Pikal, Biopharm , Sep : 18-27 (1990); J.
Pickal, Pharmaceutical Res. , 8 (4): 427-4
37 (1991)]. The glass transition increases as the protein replaces water in the preparation. Adeno-associated virus had different effects on the Tg 'of 0.5M sucrose formulations. The addition of virus at a concentration of 1.55 × 10 ¹¹ pieces of genome copies per 1ml reduced the glass transition temperature of -33.5 to -34.71 ℃.
Addition of another log of virus lowered the Tg 'to -35.37 ° C. These results suggest that this virus may interact with excipients in the frozen state differently than traditional proteins. Based on this information, the primary drying temperature of the Ad preparation was set at -35 ° C. AAV was dried at -38 ° C. Example 21-Effect of Final Moisture Content of Lyophilized Cake on Recovery of Adenovirus Various blends of sucrose and mannitol were subjected to standard freeze drying cycle (30 mt).
Final moisture content after -40 ° C. for 2 hours, −35 ° C. for 11 hours, −10 ° C. for 2 hours, 0 ° C. for 2 hours and 25 ° C. for 3 hours at orr pressure).

The water content of the lyophilized formulation was determined by Karl Fischer titration [J.
C. May et al . , Dev. Biol. Stand , 74 : 153-164 (199).
2)], and thermogravimetric analysis (TGA) [J. C. May et al . Biol. Standardization , 10 : 249-259 (1982)]. Aqua Star Karl Fischer Titrator (Model C30) equipped with a frit-free cell for Karl Fischer titration
00, EM Sciences) was calibrated with Karl Fischer water standards (2-methoxyethanol) and blanked with the addition of 1 ml absolute methanol (EM Sciences). ). The lyophilized sample was reconstituted with anhydrous methanol and added to the titrator. TGA is Perkin-Elmer (Perkin
-Elmer) TGA 7 series thermogravimetric system. Three to five milligrams of lyophilized preparation were scanned from 25 to 200 ° C at 10 ° C / min.

The data provided in Table 9 below is the average of 10 vials from a single lyophilization run.

[0096]

[Table 9]

When adenovirus was added to these preparations at a concentration of 5.12 × 10 ¹¹ lfu / ml, recovery of infectious virus was extremely sensitive to the final water content of the product (Table 9). ). The 1: 4 sucrose: mannitol preparation had a final water content of 0.6% and suffered a 1.1 log loss of virus titer. A 3: 4 ratio with a water content of 1.3% lost less than 1 log of infectious virus. Preparations consisting of a 1: 1 ratio of sucrose: mannitol did not experience any loss of potency during the entire lyophilization process. The water content of this preparation was 1.4%. When the water content exceeded this by 0.2% and rose to 1.6%, the titer dropped by 2.2 log units. These data indicate that adenovirus is not only sensitive to the final water content in the lyophilized product, but there is a relatively narrow region in which virus titer can be maintained. As a result, the entire adenovirus lyophilization step was performed to provide maximum recovery of virus of 1.3-
Specially made to yield a final moisture content of 1.5%. Example 22-Effect of virus concentration on recovery of virus titer after lyophilization To assess the maximum acceptable concentration of adenovirus that facilitates optimal recovery of virus titer after lyophilization, adenovirus was used. Lyophilized at two different concentrations in two separate formulations. The results provided in Table 10 are the average of 10 vials of each preparation.

[0098]

[Table 10]

In general, preparations with higher initial titers resulted in better recovery of active virus after lyophilization. Preparations with low virus concentrations (2-5 × 10 ¹⁰ lfu / ml) suffered approximately 1 log loss of virus regardless of formulation. Loss of titer was less than 1 log at higher adenovirus concentrations (2-5 × 10 ¹¹ lfu / ml). This may be due, in part, to the ability of the virus itself to assist in maintaining the final frozen pH of the preparation upon freezing at high concentrations (data not shown). Adenovirus preparations of 1-5 × 10 ¹¹ lfu / ml were used in additional lyophilization studies. Example 23-Stability of lyophilized viral vector After careful evaluation of the effects of process and formulation, a large scale preparation of adenovirus (1 x 10 ¹¹ lfu / ml) was prepared according to the following protocol: pressure of 30 mtorr. At -40 ° C for 2 hours, -35 ° C for 9 hours, -10 ° C for 4 hours, 0 ° C for 4 hours and 25
Lyophilized in 1M sucrose formulation at 8 ° C for 8 hours. After lyophilization, some vials were reconstituted.

The only loss detected in this preparation after 1 year at 4 ° C. was the initial loss of 0.5 log virus by the process itself. Virus from the same lot prepared in 10% glycerol and stored at -20 ° C underwent a drop in titer of approximately 2 logs over the same time period. The stability of the reconstituted preparation stored at 4 ° C was also evaluated. Viral titer is 0.5 log / over a period of 4 days
It dropped at a steady rate of the day, indicating that the vector should be used immediately after reconstitution. This degradation rate was similar for other lyophilized preparations regardless of formulation (data not shown). Studies are currently underway to elucidate the nature of this rapid drop in titer and to develop new mixed states for reconstitution that promote viral stability.

Little research has been done in developing formulations for AAV vectors and optimizing the lyophilization process. In a single experiment, an AAV preparation (8.7 x 1
Half (0 ⁸ lfu / ml) was desalted in potassium phosphate buffered saline. The remaining half was desalted to a formulation of 0.4% sucrose, 0.4% mannitol and protamine. Each preparation was subjected to the following protocol: -40 ° C. for 2 hours, −38 ° C. for 11 hours, −10 ° C. for 2 hours, 0 ° C. for 2 hours and 25 ° C. at a pressure of 30 mtorr.
Lyophilized under 3 hours.

After the initial loss of 0.3 log of titer by the freeze-drying process, the formulated preparation was 25
We did not experience any loss of infectivity for 90 days at ° C. It is also important to note that the preparations made in buffer without any additional excipients (buffers) experienced a similar loss of titer after lyophilization. This suggests that AAV may be resistant to the stress of the lyophilization process.

All publications cited in this specification are incorporated herein by reference. While the present invention has been described with respect to a particularly preferred embodiment, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

[Brief description of drawings]

FIG. 1A: Final frozen p of formulation for stability of adenovirus according to the invention.
The results of the study on the influence of H will be explained concretely (correlation coefficient, r ² = 0.98
). See Example 15. Results are reported as the average titer of 4-6 samples from 2 separate experiments. Error bars reflect the standard deviation of the data.

FIG. 1B illustrates the results of a study of the effect of the final frozen pH of a formulation on the stability of adeno-associated virus according to the invention (correlation coefficient, r ² = 0.
98). See Example 15. Results are reported as the average titer of 4-6 samples from 2 separate experiments. Error bars reflect the standard deviation of the data.

FIG. 1C: Sodium phosphate (DPBS) or potassium phosphate (KPBS) according to the invention.
The results of a -20 ° C stability study of adenoviruses prepared in either buffered saline and 10% glycerol are illustrated.

FIG. 2 illustrates the results of a study of the effect of the addition of excipients to the adenovirus preparation on the recovery of the active vector after lyophilization according to the invention. The pre-lyo titer of the whole lot was 1 × 10 ¹¹ lfu / ml. Suc /
Sucrose, mannitol and Span in Man / Span formulations 20
Are 40 mg / mL, 40 mg / mL and 0.001% respectively.
K = KPBS, D = DPBS. Data are the average of 8 vials from 2 separate experiments. Error bars represent the standard error of the data.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 47/42 A61K 47/42 47/48 47/48 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Wilson, James A. M. Pennsylvania 19035 Gladwin, USA North Avignon Drive 1350 F term (reference) 4C076 AA12 AA95 BB01 BB13 BB15 BB16 BB27 BB29 BB30 DD22 DD26 DD38 DD46 DD67 EE39 EE41 EE49 EE59 4C084 AA13 MA05 MA17 MA17 MA60 MA16 NA13 MA83 MA05A17 MA83 MA52 MA53A05 MA83 MA53A53 MA83A05 MA83A05 MA83 MA53A53 MA83A05 MA83A53 MA83MA05 MA17A03 MA83MA05 MA17A53 MA83MA05 MA17MA13 MA53MA52

Claims (23)

[Claims] 1. A) a) reacting the activated polyethylene glycol and the recombinant virus at ambient temperature for about 1 to about 2 hours; and b) stopping the reaction, whereby the polyethylene glycol bound virus. A method of conjugating a recombinant virus with polyethylene glycol to enhance its transduction efficiency, which comprises the step of: 2. The method of claim 1, wherein the activated polyethylene glycol and the recombinant virus are reacted at a polyethylene glycol to virus ratio of about 10: 1. 3. The reaction takes place in solution and the recombinant virus is 1 ml in solution.
3. The method of claim 1 or claim 2, wherein the method is present at a concentration of about 1 x 10 ^{10 to} about 1 x 10 ¹⁵ particles per. 4. The method according to claim 1, wherein the polyethylene glycol is activated with a compound selected from the group consisting of tresyl chloride, succinimidyl succinate and cyanuric chloride. 5. The method according to claim 1, wherein the polyethylene glycol has a molecular weight of 5000. 6. The method according to any one of claims 1 to 5, wherein the polyethylene glycol is monomethyoxy polyethylene glycol. 7. The recombinant virus is an adeno-associated virus.
7. The method according to any one of 6 to 6. 8. The method according to claim 1, wherein the recombinant virus is an adenovirus. 9. A virus to which polyethylene glycol is bound, which is produced by the method according to any one of claims 1 to 7. 10. The virus of claim 9, wherein the virus is selected from the group consisting of adenovirus and adeno-associated virus. 11. The virus of claim 9, wherein the virus is associated with monomethyoxy polyethylene glycol. 12. A method of increasing transduction efficiency of a recombinant virus, which comprises the step of delivering the modified recombinant virus of claim 9 to a host cell. 13. (a) The polyethylene glycol (PEG) according to claim 9.
A.) Contacting the host cell with a virus modified in), said virus comprising a molecule for delivery to the host cell; and (b) the host cell with a recombinant virus comprising said molecule. A method of re-administering a molecule via a viral vector to a selected host cell comprising the step of contacting. 14. The host cell is contacted with a PEG-modified virus after contacting the host cell with a recombinant virus comprising the molecule.
The method described in. 15. The method of claim 13, wherein the recombinant virus comprising the molecule is a second PEG-modified virus. 16. In the first PEG-modified virus and the second PEG-modified virus, PEG is monomethoxy PEG (MPEG), and MPEG is activated by different groups for each virus. 16. The method of claim 15, wherein the method is performed. 17. The MPEG is activated with a compound selected from the group consisting of tresyl chloride, succinimidyl succinate, and cyanuric chloride.
The method described in. 18. Delivery of a selected molecule to a host cell comprising a polyethylene glycol-conjugated virus produced by the method of any of claims 1-7 and a physiologically acceptable carrier. Useful compositions. 19. A recombinant viral vector comprising (a) a molecule for delivery to a host cell; (b) sucrose; and (c) mannitol (the ratio of sucrose to mannitol is about 1 to about 1). A composition that enhances the physical stability of a viral vector. 20. The composition of claim 19, wherein the composition is lyophilized to a final water content of about 1.2% to about 1.7%. 21. The composition in solution is about 1 × 1 per milliliter of solution.
20. The composition of claim 19, comprising 0 ^{10 to} about 1 x 10 ¹⁵ particles of recombinant virus. 22. The method of claim 19, further comprising β-cyclodextrin.
The composition according to. 23. The composition of claim 19, further comprising protamine.